The hemocytometer is a fundamental laboratory tool used across various scientific disciplines for precisely counting microscopic particles, such as cells, within a liquid suspension. This device, first crafted by French anatomist Louis-Charles Malassez in 1874, marked a significant advancement by establishing a standardized method for cell enumeration, which remains relevant in modern research and clinical settings. This instrument provides reliable cell counts when determining cell concentration.
Understanding the Hemocytometer
A hemocytometer is constructed from a thick piece of specialized glass, resembling a microscope slide. It features a rectangular indentation in its central area, forming a precision volume chamber. When a specially designed coverslip is placed over this chamber, a precisely known volume is created between the coverslip and the chamber’s base. This known volume is crucial for calculating the concentration of particles within a fluid sample. The chamber’s surface is engraved with a laser-etched grid of perpendicular lines, which aids in the counting process.
Dissecting the Counting Grid
The most common type of hemocytometer features an “Improved Neubauer” counting grid. This grid is composed of nine large 1 millimeter (mm) by 1 mm squares, totaling a 9 mm² area.
The four corner squares are subdivided into 16 smaller squares. The central square is further subdivided into 25 medium squares, which are then divided into 16 even smaller squares. The consistent depth of the counting chamber, precisely 0.1 mm, combined with the known dimensions of these squares, defines the exact volume above each grid area, enabling accurate counting.
The Step-by-Step Counting Process
Before counting, the cell sample often requires preparation, such as dilution, to achieve an optimal concentration for counting, usually around 10^6 cells/mL. Dilution ensures cells are adequately spaced, preventing overcrowding that could lead to counting errors. If assessing cell viability, a dye like trypan blue may be added, which stains only dead cells, allowing differentiation between live and dead cells under the microscope.
After preparation, a small volume of the sample, typically 10 microliters, is carefully loaded into the hemocytometer chamber. Capillary action draws the cell suspension evenly under the coverslip, ensuring uniform distribution without air bubbles.
Once loaded, the hemocytometer is placed under a microscope, and the grid is brought into focus, often starting at a lower magnification like 40x. Counting involves systematically moving across selected squares, such as the four large corner squares and the central square for larger cells. To maintain accuracy and avoid double-counting, a standard rule is applied: cells touching the top or left boundary lines of a square are counted, while those touching the bottom or right lines are excluded.
Unlocking Cell Concentration Formulas
After counting the cells in the designated areas of the hemocytometer, these raw counts are translated into a meaningful cell concentration per unit volume. The general formula to determine cell concentration is: (Number of cells counted / Number of squares counted) x Dilution Factor x Volume Factor.
The “Number of cells counted” is the total count from the selected squares. The “Number of squares counted” reflects the specific areas used for enumeration. The “Dilution Factor” accounts for any prior dilution of the original sample, ensuring the calculated concentration reflects the undiluted state.
The “Volume Factor” converts the counted cells within the hemocytometer’s small volume into a standard unit, typically cells per milliliter (cells/mL). For a standard Improved Neubauer chamber, each large square, with a depth of 0.1 mm, represents a volume of 0.1 mm³. The volume factor is commonly 10^4. The total number of cells counted is multiplied by this factor to yield the cell concentration per milliliter.
Where Hemocytometers are Used
Hemocytometers are widely employed across various fields:
In clinical diagnostics, for counting blood cells, including red and white blood cells, which can indicate health conditions like anemia or infection.
In reproductive biology, for sperm counts.
In biotechnology and cell culture laboratories, to monitor cell growth, determine cell viability, and prepare cell suspensions for further experiments.
In the food and beverage industry, for counting yeast cells in brewing processes.